Questions tagged [rna-seq]

Questions should include this tag if they pertain to issues related to bioinformatics analysis of RNA-seq data, e.g. normalization, differential expression analysis, sequencing and experimental design or transcriptome assembly...

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12 views

How to perform a meta-analysis using data consisting of paired-end and single-end reads generated from Illumina and Ion Torrent?

So basically I have RNA-seq reads that were generated from Illumina and Ion Torrent platforms for yeast species. I have seen an article where they compared liver cells of a rat that were sequenced ...
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2answers
35 views

Is it possible to do DEG analysis without replicates?

I have three bulk RNA-seq samples (test1, test2, test3) without replicates. And I noticed that DEG analysis tools such as DESeq2/edgeR/etc cannot be applied for data with no replicates. So, I just ...
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3answers
481 views

RNAseq biological replicates not clustering in PCA plots

I have RNAseq data from 4 samples with 3 biological replicates per sample. I am currently trying to do the differential expression analysis with DESeq2 but the biological replicates will not cluster ...
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136 views

Negative binomial modeling of RNA-Seq data

A common way to model RNA-seq data is using a negative binomial distribution, where each sample-gene pair is modeled by a different negative binomial distribution with mean $\mu_{ij}$ where $i$ and $j$...
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1answer
26 views

Assembling all transcripts for an individual gene? (using single sequence to seed the assembly)

Let's say I have a candidate gene and I believe that in an individual sample, the genome sequence differs from the reference which then interferes with alignment. Is there a way for me to do a "...
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1answer
428 views

insert size pre and post trimming

I have a problem here with my rna seq data: Sequencing details: rRNA was removed, followed by cDNA preparation and generation of stranded libraries using the TruSeq Stranded Total RNA Sample Prep Kit. ...
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10 views

java error when running CIRI-full.jar Merge module

I have an issue running a module trying to detect circRNA from RNAseq data. The error is a java error and I have not knowledge of java so I don't understand the issues and googling hasn't helped. the ...
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16 views

A very large number of clones in BCR reportorie

I am using mixcr to convert fq.gz raw data file (single cell BCR sequencing) to txt files with the names such as JX01_d3-B.clonotypes.IGH.txt , Then I use the immunarch to load the file to explore the ...
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1answer
29 views

Parallelizing microRNA targets

I am trying to look for miRNA targets using a file called Conservedfamily.txt from the Zebrafish target scan fish website. I have written a python program to ...
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2answers
41 views

Counting the number of gene isoforms in a GFF3, is this method correct?

Recently I've been tasked to count the numbers of gene isoforms for each locus in a .gff3 file. I'm still doing my first steps in biology and bioinformatics, so I ...
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1answer
27 views

Error when using awk command to trim sequence files

I am attempting to edit my fastq sequence files with an awk command from a published article. The sequence files are all ...
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44 views

Splitting .bam files into separate samples for tophat2

I have RNA seq data of 12 samples (paired end, so 24 files) and I ran tophat2 for alignment on all of them which gave me one bam file for all the samples. Now I need separate bam files for all the ...
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1answer
32 views

Limma decideTests function: what kind of multiple hypothesis testing correction does parameter "method" involve?

What kind of multiple hypothesis testing correction does method="global" do in Limma's decideTests function? According ...
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1answer
56 views

Determining what RNAseq data is filtered on volcano plot

I am using RNA seq data to analyze genes via a volcano plot comparing differential gene expression of bacteria with and without antibiotic in R. After having created my plot, I am unsure why some of ...
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1answer
162 views

"perl: warning: Setting locale failed." in RepeatMasker

I'm trying to run Repeatmasker in Linux on the command line with: ...
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3answers
78 views

Explanation for RNA-seq samples not clustering in PCA as expected

A colleague is analysing RNA-seq data - the study design is 2 treatments, 3 replicates, 3 tissues. In their PCA plot the samples clustered neatly by tissue. Except for two samples - two tissue samples ...
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19 views

Gene co-expression network analysis

I am working on a genome-wide study and struggling with gene co-expression network analysis. I am working on Tomato(Solanum lycopersicum). Can someone suggest me some tools or methods for this ...
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1answer
27 views

Keeping rows with a cut-off in all columns

I have a data frame of raw read counts, genes in rows and samples in columns ...
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1answer
198 views

hisat2 --rna-strandness option and downstream htseq-count analysis

I've got some doubts on the hisat2 --rna-strandness option and its output for downstream analysis. Is it expected to see a difference in alignment and counting ...
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4answers
2k views

Convert R RNA-seq data object to a Python object

I have done some work in R and would like to try a Python tool. What is a good way to import the data (and its annotations etc) as a Python object? I am particularly interested in converting a ...
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1answer
55 views

Beginner with DESeq2 having issue with analysis

Hi there I am brand new to using RStudio and trying to run DESeq2 analysis on my featureCounts output counts table. I ran the following code: ...
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1answer
39 views

shiny dashboard to visualise RNA seq data

I am trying to build a shiny dashboard for the visualization of different RNAseq data sets. However, I am encountering problem with reactive input, the code is always using the second dataset that I ...
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1answer
475 views

lower mapping rates in salmon v0.13 compared to previous versions

Hi there :) Thanks for the tool! I recently updated to the new salmon (from 0.8... its been a couple years) and I noticed that my mapping percentages change dramatically between the two versions. For ...
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2answers
85 views

Calculate genome coverage and depth from alignment

I have a .bam alignment file and a genome reference .fasta file. I am looking for a easy to use tool (that I can reference in a publication) to calculate the percentage coverage of the reference by ...
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1answer
44 views

DESeq2. Which is better: using the altHypothesis argument in the results function or manually filtering for your desired P-value and Log2FoldChange?

In DESeq2 you can identify your differentially expressed (DE) genes using the results function. I noticed people in my lab using the results() function with the minimum number of arguments supplied (...
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1answer
46 views

Table error when combining counts columns

I have these files that I want to make table of (https://github.com/learnseq/RNAseqfiles01.git), the table that I want (since all the files have the same ID column),, I want merge the ID column with ...
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1answer
202 views

CIBERSORT runtime error eval failed

I was running CIBERSORT but caught this error right after it started permutation: ...
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0answers
31 views

How to detail the specific GO terms

How can I get more specific GO terms when using clusterProfiler? I got my Dotplot by: ...
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0answers
229 views

How to convert Illumina IDs to Gene Names?

I am new to the area of bioinformatics, so apologies if this is too obvious of a query. I need to analyze the RNA Seq data from GSE98455. The dataset is of the following format: ...
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3answers
79 views

The confusion of using TPM (transcripts per million)

It is shown that TPM values are not suitable for DEG analysis but good for within-sample comparison since TPM normalized the gene length. My question is first: if TPM is not suitable for across ...
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1answer
46 views

Understanding "Measurement of mRNA abundance using RNA-seq data" by Wagner et al 2012

I'm trying to understand why RPKM is not an appropriate way to normalize for RNA seq (I understand the general idea, but I'd like to gain a deeper understanding). So I'm reading the original paper by ...
2
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1answer
68 views

Comparing multiple treatments to multiple other treatments in edgeR for simple effects in a complex experimental design

I am working with a RNA-seq data set in maize that has a relatively complex design. There are two levels of treatment A (nitrogen fertilizer level in the field, high or low), two levels of treatment B ...
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3answers
76 views

What is a good RNA seq normalization method that allows for across sample comparisons and between transcripts

What is a good RNA seq normalization method that allows for across sample comparisons, and allows between transcripts comparisons as well? I read that TMM for example allows across sample comparisons ...
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1answer
34 views

What is a good rule of thumb for the threshold of noise versus signal for RPK in RNA seq?

I have RPK values (RNA seq) and I'm wondering what is a good rule of thumb for what is considered to be noise versus what is considered to be signal? I.e what should I choose as a threshold value for ...
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0answers
229 views

R package equivalent to RSeQC infer_experiment to get strandedness of RNA-Seq

I am currently writing an R package that includes a module to run featureCounts (gene quantification tool) from Rsubread. I wanted to be able to specify the correct strandedness option to ...
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1answer
104 views

Which module to select for Pathway analysis based on Module trait correlation and pvalue?

I have a total of 35 tumor samples classified into 4 subtypes. Subtype A, B, C, and D. I have RNAseq data. I'm interested in identifying modules related to each subtype with co-expression network ...
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117 views

Run cuffcompare in strand-agnostic mode

Is there a way to run Cufflinks' cuffcompare in a strand-agnostic mode? I would like to do this because I have some RNA-seq datasets derived from an unstranded run, that should be compared to a ...
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0answers
8 views

Taiji doesn't run Pagerank

I'm trying to use Taiji to run a pagerank on our combined bulk RNAseq and ATACseq dataset. Here's how we run it: taiji run --config config.yml -n 10 Here's ...
3
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1answer
161 views

sva for RNA-Seq data without known phenotype

I have been working on RNA-Seq data from two different cohorts, and they show very strong batch effect (~35% variance explained by 1st component in PCA). Since I am trying to do a class discovery from ...
3
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0answers
111 views

How do I resolve disagreements in the determination of significant genes?

I want to find some good predictors (genes). I have checked the Spearman correlation of the expression of each of 23 genes with dependent variables (responders Vs non-responders and I saw only the 5 ...
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1answer
42 views

how to get normcounts for singlecellexperiment object?

I need to perform differential expression analysis using the scDD package from R, but I am not able to since I miss the normcounts assay in my SCE object (of course in the example they show, the assay ...
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1answer
83 views

How to calculate module-trait relationship when trait data is in binary format?

I have a dataset of 50 breast cancer samples. These samples are classified into four subtypes Lum A, Lum B, Her2 and Basal. I have been working with lncRNAs and protein-coding genes. To identify the ...
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1answer
55 views

Batch effect correction using a subset of samples - using DE genes

I have some RNA-seq data with two very obvious batches as you can see in the PCA: The samples of interest (A - H) are from tumor tissues. In addition, there are data for two cell lines I and J. The ...
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2answers
167 views

Problem with merge data while trying to convert gene names in R

This question has also been asked on Biostars and StackOverflow I've been trying to code (in R) a way to convert gene accession numbers to gene names (from RNAseq data). I've looked at all the related ...
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37 views

Correcting for batch effect in bulk RNA seq datasets

I have samples from many different bulk RNA seq studies that exhibit an evident batch effect (samples from different labs cluster together regardless of treatment). I've found a lot of papers (here, ...
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1answer
75 views

RNA_Seq Analysis in R, propmapped function issue

I'm currently trying to learn RNA-seq data analysis and differential expression in R. I've been using the Combine lab tutorial. Everything was running well until the tutorial asked to use the ...
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1answer
31 views

Filtering genes from cuffdiff results

This question has also been asked on Biostars I have run cuffdiff (with statistics turned ON) to compare two groups of samples: Control group and Late AD group. This is the command I ran to be precise:...
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0answers
29 views

How to create a DESeqDataSet and define experiment design before varianceStabilizingTransformation?

I have an RNAseq count matrix consisting of 2 groups (high, low) with 6 timepoints per group (T1,T2,...,T6) and 3 replicates per timepoint (rep1, rep2, rep3). So a 2-factor design with 36 samples in ...
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1answer
16 views

Combining read counts from three separate GEO studies

I want to do differential expression analysis with DESEQ2. I have three read counts files downloaded from GEO (small RNAseq based) where the number of miRNAs and id is nearly the same. These studies ...
2
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1answer
109 views

How to visualize genome track of gene in specific cell-lines?

I'm trying to make a plot showing genome tracks of specific genes in specific cell-lines of RNA-seq and Chip-seq data. It should look something like this I have recently seen this encode, but in ...

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